Genetically engineered plant cells and plants exhibiting resistance to glutamine synthetase inhibitors, DNA fragments and recombinants for use in the production of said cells and plants

ABSTRACT

The invention relates to a DNA fragment containing a determined gene, the expression of which inhibits the antibiotic and herbicidal effects of Bialaphos and related products. 
     It also relates to recombinant vectors, containing such DNA fragment, which enable this protective gene to be introduced and expressed into cells and plant cells.

This application is a divisional of application Ser. No. 07/525,300,filed May 17, 1990, now U.S. Pat. No. 5,561,236, which is a continuationof application Ser. No. 07/131,140, filed Nov. 5, 1987, abandoned, whichis a continuation under 35 USC §§363 and 120 of PCT/EP 87/00141, filedMar. 11, 1987, published as WO 87/05629.

The invention relates to a process for protecting plant cells and plantsagainst the action of glutamine synthetase inhibitors.

It also relates to applications of such process, particularly to thedevelopment of herbicide resistance into determined plants.

It relates further to non-biologically transformed plant cells andplants displaying resistance to glutamine synthetase inhibitors as wellas to suitable DNA fragments and recombinants containing nucleotidesequences encoding resistance to glutamine synthetase inhibitors.

Glutamine synthetase (hereafter simply designated by GS) constitutes inmost plants one of the essential enzymes for the development and life ofplant cells. It is known that GS converts glutamate into glutamine. GSis involved in an efficient pathway (the only one known nowadays) inmost plants for the detoxification of ammonia released by nitratereduction, aminoacid degradation or photorespiration. Therefore potentinhibitors of GS are very toxic to plant cells. A particular class ofherbicides has been developped, based on the toxic effect due to inhibitinhibition of GS in plants.

These herbicides comprise as active ingredient a GS inhibitor.

There are at least two possible ways which might lead to plantsresistant to the inhibitors of the action of glutamine synthetase; (1)by changing the target. It can be envisaged that mutations in the GSenzyme can lead to insensitivity towards the herbicide; (2) byinactivation of the herbicide. Breakdown or modification of theherbicide inside the plant could lead to resistance.

Bialaphos and phosphinothricin (hereafter simply designated by PPT) aretwo such inhibitors of the action of GS, (ref. 16, 17) and have beenshown to possess excellent herbicidal properties (see more particularlyref. 2 as concerns Bialaphos).

Bialaphos has the following formula (I)

PPT has the following formula (II)

Thus the structural difference between PPT and Bialaphos resides in theabsence of two alanine aminoacids in the case of PPT.

These two herbicides are non selective. They inhibit growth of all thedifferent species of plants present on the soil, accordingly cause theirtotal destruction.

Bialaphos was first disclosed as having antibiotic properties, whichenabled it to be used as a pesticide or a fungicide. Bialaphos can beproduced according to the process disclosed in U.S. Pat. No. 3,832,394,assigned to MEIJI SEIKA KAISHA LTD., which patent is incorporated hereinby reference. It comprises cultivating Streptomyces hygroscopicus, suchas the strain available at the American Type Culture Collection, underthe ATCC number 21,705, and recovering Bialaphos from its culturemedium. However, other strains, such as Streptomyces viridochromogenes,also produce this compound (ref. 1).

Other tripeptide antibiotics which contain a PPT moiety are or might bediscovered in nature as well, e.g. phosalacin (ref. 15).

PPT is also obtained by chemical synthesis and is commerciallydistributed by the industrial Company HOECHST.

A number of Streptomyces species have been disclosed which producehighly active antibiotics which are known to incapacitate procaryoticcell functions or enzymes. The Streptomyces species which produce theseantibiotics would themselves be destroyed if they had not a self defencemechanism against these antibiotics. This self defence mechanism hasbeen found in several instances to comprise an enzyme capable ofinhibiting the antibiotic effect, thus of avoiding autotoxicity for theStreptomyces species concerned. This modification is generally reversedwhen the molecule is exported from the cell.

The existence of a gene which encodes an enzyme able to modify theantibiotic so as to inhibit the antibiotic effect against the host hasbeen demonstrated in several Streptomyces producing antibiotics, forexample, in S. fradiae, S. azureus, S. vinaceus, S. erythreus, producingneomycin, thiostrepton, viomycin, and MLS (Macrolide LincosamideStreptogramin) antibiotics respectively (ref. 4), (ref. 5), (ref.6),(ref. 14 by CHATER et al., 1982 describes standard techniques whichcan be used for bringing these effects to light).

In accordance with the present invention, it has been found thatStreptomyces hygroscopicus ATCC 21,705, also possesses a gene encodingan enzyme responsible of the inactivation of the antibiotic propertiesof Bialaphos. Experiments carried out by the applicants have lead to theisolation of such a gene and its use in a process for controlling theaction of GS inhibitors, based on PPT or derived products.

An object of the invention is to provide a new process for controllingthe action in plant cells and plants of GS inhibitors.

Another object of the invention is to provide DNA fragments and DNArecombinants, particularly modified vectors containing said DNAfragments, which DNA fragments contain nucleotide sequences capable,when incorporated in plant cells and plants, to protect them against theaction of GS inhibitors.

A further object of the invention is to provide non-biologicallytransformed plant cells and plants capable of neutralizing orinactivating GS inhibitors.

A further object of the invention is to provide a process forselectively protecting plant species against herbicides of a GSinhibitor type.

More specifically an object of the invention is to provide a DNAfragment transferable to plant cells- and to whole plants—capable ofprotecting them against the herbicidal effects of Bialaphos and ofstructurally analogous herbicides.

A further object of the invention is to provide plant cells resistant tothe products of the class examplified by Bialaphos, which productspossess the PPT unit in their structure.

The process according to the invention for controlling the action inplant cells and plants of a GS inhibitor when contacted therewith,comprises providing said plants with a heterologous DNA fragmentincluding a foreign nucleotide sequence, capable of being expressed inthe form of a protein in said plant cells and plants, under conditionsuch as to cause said heterologus DNA fragment to be integrated stablythrough generations in the cells of said plants, and wherein saidprotein has an enzymatic activity capable of inactivating orneutralization of said glutamine synthetase inhibitor.

A preferred DNA fragment is one derived from anantibiotic-producing-Streptomyces strain (or a sequence comprising anucleotide sequence encoding the same activity) and which encodesresistance to a said GS inhibitors.

Preferred nucleotide sequences for use in this invention encode aprotein which has acetyl tranferase activity with respect to said GSinhibitors.

A most preferred DNA fragment according to the invention comprises anucleotide sequence coding for a polypeptide having a PPT acetyltransferase activity.

A particular DNA fragment according to the invention, for the subsequenttransformation of plant cells, consists of a nucleotide sequence codingfor at least part of a polypeptide having the following sequence (SEQ IDNO:1):

                                             X SER PRO GLU   183ARG ARG PRO ALA ASP ILE ARG ARG ALA THR GLU ALA ASP MET PRO   228ALA VAL CYS THR ILE VAL ASN HIS TYR ILE GLU THR SER THR VAL   273ASN PHE ARG THR GLU PRO GLN GLU PRO GLN GLU TRP THR ASP ASP   318LEU VAL ARG LEU ARG GLU ARG TYR PRO TRP LEU VAL ALA GLU VAL   363ASP GLY GLU VAL ALA GLY ILE ALA TYR ALA GLY PRO TRP LYS ALA   408ARG ASN ALA TYR ASP TRP THR ALA GLU SER THR VAL TYR VAL SER   453PRO ARG HIS GLN ARG THR GLY LEU GLY SER THR LEU TYR THR HIS   498LEU LEU LYS SER LEU GLU ALA GLN GLY PHE LYS SER VAL VAL ALA   543VAL ILE GLY LEU PRO ASN ASP PRO SER VAL ARG MET HIS GLU ALA   588LEU GLY TYR ALA PRO ARG GLY MET LEU ARG ALA ALA GLY PHE LYS   633HIS GLY ASN TRP HIS ASP VAL GLY PHE TRP GLN LEU ASP PHE SER   678LEU PRO VAL PRO PRO ARG PRO VAL LEU PRO VAL THR GLU ILE   723in which X represents MET or VAL, which part of said polypeptide is ofsufficient length to confer protection against Bialaphos to plant cells,when incorportated genetically and expressed therein, i.e. as termedhereafter “plant-protecting capability” against Bialaphos.

A preferred DNA fragment consists of the following nucleotide sequence(SEQ ID NO:2):

                                            GTG AGC CCA GAA   183CGA CGC CCG GCC GAC ATC CGC CGT GCC ACC GAG GCG GAC ATG CCG   228GCG GTC TGC ACC ATC GTC AAC CAC TAC ATC GAG ACA AGC ACG GTC   273AAC TTC CGT ACC GAG CCG CAG GAA CCG CAG GAG TGG ACG GAC GAC   318CTC GTC CGT CTG CGG GAG CGC TAT CCC TGG CTC GTC GCC GAG GTG   363GAC GGC GAG GTC GCC GGC ATC GCC TAC GCG GGC CCC TGG AAG GCA   408CGC AAC GCC TAC GAC TGG ACG GCC GAG TCG ACC GTG TAC GTC TCC   453CCC CGC CAC CAG CGG ACG GGA CTG GGC TCC ACG CTC TAC ACC CAC   498CTG CTG AAG TCC CTG GAG GCA CAG GGC TTC AAG AGC GTG GTC GCT   543GTC ATC GGG CTG CCC AAC GAC CCG AGC GTG CGC ATG CAC GAG GCG   588CTC GGA TAT GCC CCC CGC GGC ATG CTG CGG GCG GCC GGC TTC AAG   633CAC GGG AAC TGG CAT GAC GTG GGT TTC TGG CAG CTG GAC TTC AGC   678CTG CCG GTA CCG CCC CGT CCG GTC CTG CCC GTC ACC GAG ATC   723or of a part thereof expressing a polypeptide having plant-protectingcapability against Bialaphos.

The invention also relates to any DNA fragment differing from thepreferred one indicated hereabove by the replacement of any of itsnucleotides by others, yet without modifying the genetic information ofthe preferred DNA sequence mentioned hereabove (normally within themeaning of the universal genetic code), and furthermore to anyequivalent DNA sequence which would encode a polypeptide having the sameproperties, particularly a Bialaphos-resistance-activity.

It will be understood that the man skilled in the art should be capableof readily assessing those parts of the nucleotide sequences that couldbe removed from either side of any of the DNA fragments according to theinvention, for instance by removing terminal parts on either side ofsaid DNA fragment, such as by an exonucleolytic enzyme, for instanceBal31, by recloning the remaining fragment in a suitable plasmid and byassaying the capacity of the modified plasmid to transform appropriatecells and to protect it against the Bialaphos antibiotic or herbicide asdisclosed later, whichever assay is appropriate.

For the easiness of language, these DNA fragments will be termedhereafter as “Bialaphos-resistance DNA”. In a similar manner, thecorresponding polypeptide will be termed as “Bialaphos-resistanceenzyme”.

While in the preceding discussion particular emphasis has been put onDNA fragments capable, when introduced into plant cells and plants, toconfer on them protection against Bialaphos or PPT, it should beunderstood that the invention should in no way be deemed as limitedthereto.

In a same manner, the invention pertains to DNA fragments which, whenintroduced into such plant cells, would also confer on them a protectionagainst other GS inhibitors, for instance of intermediate productsinvolved in the natural biosynthesis of phosphinotricin, such as thecompounds designated by the abbreviations MP101 (III), MP102 (IV), theformula of which are indicated hereafter:

More generally, the invention has opened the route to the production ofDNA fragments which, upon proper incorporation into plant cells andplants, can protect them against GS inhibitors when contacted therewith,as this will be shown in a detailed manner in relation to Bialaphos andPPT in the examples which will follow.

This having been established, it will be appreciated that any fragmentencoding an enzymatic activity which would protect plant cells andplants against said GS inhibitors, by inactivationg, should be viewed asan equivalent of the preferred fragments which have been disclosedhereabove. This would apply especially to any DNA fragments that wouldresult from genetic screening of the genomic DNAs of strains,particularly of antibiotic-producing strains, likely to possess geneswhich, even-though structurally different, would encode similar activitywith respect to Bialaphos or PPT, or even with respect to other GSinhibitors. This applies to any gene in other strains producing a PPTderivative.

Therefore, it should be understood that the language“Bialaphos-resistance DNA” or “Bialaphos-resistance enzyme” usedthereafter as a matter of convenience is intended to relate not only tothe DNAs and enzymes specifically concerned with resistance to PPT ormost directly related derivatives, but more generally with other DNAsand enzymes which would be capable, under the same circumstances, ofinactivating the action in plants of GS inhibitors.

The invention also relates to DNA recombinants containing the abovedefined Bialaphos-resistance DNA fragments recombined with heterologousDNA, said heterologous DNA containing regulation elements and saidBialaphos-resistance DNA being under the control of said regulationelements in such manner as to be expressible in a foreign cellularenvironment compatible with said regulation elements. Particularly theabovesaid Bialaphos-resistance-DNA fragments contained in said DNArecombinants are devoid of any DNA region involved in the biosynthesisof Bialaphos, when said Bialaphos-resistance-DNA fragment originatethemselves from Bialaphos-producing strains.

By “heterologous DNA” is meant a DNA of an other origin than that fromwhich said Bialaphos-resistance-DNA originated, e.g. is different fromthat of a Streptomyces hygroscopicus or Streptomyces viridochromogenesor even more preferably a DNA foreign to Streptomyces DNA. Particularlysaid regulation elements are those which are capable of controlling thetranscription and translation of DNA sequences normally associated withthem in said foreign environment. “Cellular” refers both tomicroorganisms and to cell cultures.

This heterologous DNA may be a bacterial DNA, particularly when it isdesired to produce a large amount of the recombinant DNA, such as foramplification purposes. In that respect a preferred heterologous DNAconsists of DNA of E. coli or of DNA compatible with E. coli. It may beDNA of the same origin as that of the cells concerned or other DNA, forinstance viral or plasmidic DNA known as capable of replicating in thecells concerned.

Preferred recombinant DNA contains heterologous DNA compatible withplant cells, particularly Ti-plasmid DNA.

Particularly preferred recombinants are those which contain GS inhibitorinactivating DNA under the control of a promoter recognized by plantcells, particularly those plant cells on which inactivation of GSinhibitors is to be conferred.

Preferred recombinants according to the invention further relate tomodified vectors, particularly plasmids, containing saidGS-inhibitor-inactivating DNA so positioned with respect to regulationelements, including particularly promoter elements, that they enablesaid GS inhibitor-inactivating DNA to be transcribed and translated inthe cellular environment which is compatible with said heterologous DNA.Advantageous vectors are those so engineered as to cause stableincorporation of said GS inhibitor inactivating DNA in foreign cells,particularly in their genomic DNA. Preferred modified vectors are thosewhich enable the stable transformation of plant cells and which conferto the corresponding cells, the capability of inactivating GSinhibitors.

It seems that, as described later, the initiation codon of theBialaphos-resistance-gene of the Streptomyces hygroscopicus strain usedherein is a GTG codon. But in preferred recombinant DNAs or vectors, theBialaphos-resistance-gene is modified by substitution of an ATGinitiation codon for the initiation codon GTG, which ATG enablestranslation initiation in plant cells.

In the example which follows, the plant promoter sequence which has beenused was constituted by a promoter of the 35 S cauliflower mosaic virus.Needless to say that the man skilled in the art will be capable ofselecting other plant promoters, when more appropriate in relation tothe plant species concerned.

According to an other preferred embodiment of the invention,particularly when it is desired to achieve transport of the enzymeencoded by the Bialaphos-resistance-DNA into the chloroplasts, theheterologous DNA fragment is fused to a gene or DNA fragment encoding atransit peptide, said last mentioned fragment being then intercalatedbetween the GS inhibitor inactivating gene and the plant promoterselected.

As concerns means capable of achieving such constructions, reference canbe made to the following British applications 84 32757 filed on Dec. 28,1984 and 85 00336 filed on Jan. 7, 1985 and to the related applicationsfiled in the United-States of America (No. 06/755,173, filed on Jul. 15,1985), in the European Patent Office (No. 85 402596.2, filed on Dec. 20,1985), in Japan (No. 299 730, filed on Dec. 27, 1985), in Israel (No. 77466 filed on Dec. 27, 1985) and in Australia (No. 5 165 485, filed onDec. 24, 1985), all of which are incorporated herein by reference.

Reference can also be made to the scientific literature, particularly tothe following articles:

VAN DEN BROECK et al., 1985, Nature, 313, 358-363;

SCHREIER and al., Embo. J., vol. 4, No. 1, 25-32.

These articles are also incorporated herein by reference.

For the sake of the record, be it recalled here that under theexpression “transit peptide”, one refers to a polypeptide fragment whichis normally associated with a chloroplast protein or a chloroplastprotein sub-unit in a precursor protein encoded by plant cell nuclearDNA. The transit peptide then separates from the chloroplast protein oris proteolitically removed, during the translocation process of thelatter protein into the chloroplasts. Examples of suitable transitpeptides are those associated with the small subunit of ribulose-1,5biphosphate (RuBP) carboxylase or that associated with the chlorophyla/b binding proteins.

There is thus provided DNA fragments and DNA recombinants which aresuitable for use in the process defined hereafter.

More particularly the invention also relates to a process, which can begenerally defined as a process for producing plants and reproductionmaterial of said plants including a heterologous genetic material stablyintegrated therein and capable of being expressed in said plants orreproduction material in the form of a protein capable of inactivatingor neutralizing the activity of a glutamine synthetase-inhibitor,comprising the non biological steps of producing plants cells or planttissue including said heterologous genetic material from starting plantcells or plant tissue not able to express that inhibiting orneutralizing activity, regenerating plants or reproduction material ofsaid plants or both from said plant cells or plant tissue including saidgenetic material and, optionally, biologically replicating said lastmentioned plants or reproduction material or both, wherein saidnon-biological steps of producing said plant cells or plant tissueincluding said heterologous genetic material, comprises transformingsaid starting plant cells or plant tissue with a DNA-recombinantcontaining a nucleotide sequence encoding said protein, as well as theregulatory elements selected among those which are capable of enablingthe expression of said nucleotide sequence in said plant cells or planttissue, and to cause the stable integration of said nucleotide sequencein said plant cells and tissue, as well as in the plant and reproductionmaterial processed therefrom throughout generations.

The invention also relates to the cell cultures containingBialaphos-resistance-DNA, or more generally saidGS-inhibitor-inactivating DNA, which cell cultures have the property ofbeing resistant to a composition containing a GS inhibitor, whencultured in a medium containing a such composition at dosages whichwould be destructive for non transformed cells.

The invention concerns more particularly those plant cells or cellcultures in which the Bialaphos-resistance DNA is stably integrated andwhich remains present over successive generations of said plant cells.Thus the resistance to a GS inhibitor, more particularly Bialaphos orPPT, can also be considered as a way of characterizing the plant cellsof this invention.

Optionally one may also resort to hybridization experiments between thegenomic DNA obtained from said plant cells with a probe containing a GSinhibitor inactivating DNA sequence.

More generally the invention relates to plant cells, reproductionmaterial, particularly seeds, as well as plants containing a foreign orheterologous DNA fragment stably integrated in their respective genomicDNAs, said fragments being transferred throughout generations of suchplant cells, reproduction material, seeds and plants, wherein said DNAfragment encodes a protein inducing a non-variety-specific enzymaticactivity capable of inactivating or neutralizing GS inhibitors,particularly Bialaphos and PPT, more particularly to confer on saidplant cells, reproduction material, seeds and plants a correspondingnon-variety-specific phenotype of resistance to GS inhibitors.

“Non-variety-specific” enzymatic activity or phenotype aims at referringto the fact that they are not characteristic of specific plant genes orspecies as this will be illustrated in a non-limitative way by theexamples which will follow. They are induced in said plant materials byessentially non-biological processes applicable to plants belonging tospecies normally unrelated with one another and comprising theincorporation into said plant material of heterologous DNA, e.g.bacterial DNA or chemically synthesized DNA, which does not normallyoccur in said plant material or which normally cannot be incorporatedtherein by natural breeding processes, and which yet confers a commonphenotype (e.g. herbicide resistance) to them.

The invention is of particular advantageous use in processes forprotecting field-cultivated plant species against weeds, which processescomprise the step of treating the field with an herbicide, e.g.Bialaphos or PPT in a dosage effective to kill said weeds, wherein thecultivated plant species then contains in their genome a DNA fragmentencoding a protein having an enzymatic activity capable of neutralizingor inactivating said GS inhibitor.

By way of illustration only, effective doses for use in the abovesaidprocess range from about 0.4 to about 1.6 kg/Hectare of Bialaphos orPPT.

There follows now a disclosure of how the preferred DNA fragmentdescribed hereabove was isolated starting from the Streptomyceshygroscopicus strain available at the American Type Culture Collectionunder deposition number ATCC 21 705, by way of exemplification only.

The following disclosure also provides the technique which can beapplied to other strains producing compounds with a PPT moiety.

The disclosure will then be completed with the description of theinsertion of a preferred DNA fragment conferring to the transformedcells the capability of inactivating Bialaphos and PPT. Thus theBialaphos-inactivating-DNA fragment designated thereafter byBialaphos-resistance gene or “sfr” gene, isolated by the above describedtechnique into plasmids which can be used for transforming plant cellsand conferring to them a resistance against Bialaphos, also merely byway of example for non-limitative illustration purposes.

The following disclosure is made with reference to the drawings inwhich:

FIG. 1 is a restriction map of a plasmid containing a Streptomyceshygroscopicus DNA fragment encoding Bialaphos-resistance, which plasmid,designated hereafter as pBG1 has been constructed according to thedisclosure which follows;

FIG. 2 shows the nucleotide sequence (SEQ ID NO:12) of a smallerfragment obtained from pBG1, subcloned into another plasmid (pBG39) andcontaining the resistance gene;

FIG. 3 shows the construction of a series of plasmids given by way ofexample, which plasmids aim at providing suitable adaptation means forthe insertion therein of the Bialaphos-resistance gene or “sfr” gene;

FIGS. 4A and 4B show the construction of a series of plasmids given byway of example, which plasmids contain suitable plant cell promotersequences able to initiate transcription and expression of the foreigngene inserted under their control into said plasmids;

FIG. 5A shows a determined fragment of the nucleotide sequence (SEQ IDNO:13) of the plasmid obtained in FIG. 3;

FIG. 5B shows the reconstruction of the first codons of aBialaphos-resistance gene, from a FokI/BglII fragment obtained frompBG39 and the substitution of an ATG initiation codon for the GTGinitiation codon of the natural “sfr” gene (SEQ ID NO:14);

FIG. 5C shows the reconstruction of the entire “sfr” gene, namely thelast codons thereof (SEQ ID NO:15), and its insertion into a plasmidobtained in FIGS. 4A and 4B;

FIG. 6A shows an expression vector containing the “sfr” gene placedunder the control of a plant cell promoter;

FIG. 6B shows another expression vector deriving from the one shown inFIG. 6A, by the substitution of some nucleotides.

FIG. 7 shows the construction of a series of plasmids given by way ofexamples, to ultimately produce plasmids containing the promoter regionand the transit peptide sequence of a determined plant cell gene, forthe insertion of the “sfr” gene under the control of said promoterregion and downstream of said transit peptide sequence (SEQ ID NOS: 16and 17).

FIGS. 8 to 11 will be referred to hereafter.

The following experiment was set up to isolate aBialaphos-resistance-gene from S. hygroscopicus, according to standardtechniques for cloning into Streptomyces.

2.5 μg of S. hygroscopicus genomic DNA and 0.5 μg of Streptomyces vectorpIJ61 were cleaved with PstI according to the method described in ref.6. The vector, fragments and genomic fragments were mixed and ligated (4hours at 10° C. followed by 72 hours at 4° C. in ligation salts whichcontain 66 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10 mM MgCl₂, 10 mM2-mercaptoethanol and 0.1 mM ATP) at a total DNA concentration of 40 μgml⁻¹ with T4 DNA ligase. Ligation products were introduced into 3×10⁹ S.lividans strain 66 protoplasts by a transformation procedure mediated bypolyethylene-glycol (PEG) as described hereafter. These protoplasts gaverise to 5×10⁷ colonies and 4×10⁴ pocks after regeneration on 20 platesof R2 agar containing 0.5% of Difco yeast extract (R2 YE). Preparationand composition of the different mediums and buffers used in thedisclosed experiments are described hereinafter. When these lawns werereplica-plated on minimal medium plates containing 50 μg ml⁻¹ Bialaphos,drug resistant colonies appeared at a frequency of 1 per 10⁴transformants. After purification of the drug resistant colonies, thereplasmid DNA was isolated and used to retransform S. lividansprotoplasts. Non selective regeneration followed by replication toBialaphos-containing-medium demonstrated a 100% correlation betweenpocks and Bialaphos resistant growth. The recombinant plasmids ofseveral resistant clones all contained a 1.7 Kb PstI insert (see FIG.1).

Subcloning of the Herbicide Resistance Gene

The 1.7 Kb PstI insert was then subcloned into the high copy numberstreptomycete vector pIJ385 to generate plasmid pBG20. S. lividansstrains which contained pBG20 were more than 500 times more resistant toBialaphos. S. lividans growth is normally inhibited in minimal mediumcontaining 1 μg/ml Bialaphos; growth of transformants containing pBG20was not noticeably inhibited in a medium containing 500 μg/ml Bialaphos.The PstI fragment was also subcloned in either orientation into the PstIsite of the plasmid pBR322, to produce plasmids pBG1 and pBG2, accordingto their orientation. A test on minimal M9 medium demonstrated that E.coli E8767 containing pBG1 or pBG2 was resistant to Bialaphos.

A ±1.65 Kb PstI-BamHI fragment was subcloned from pBG1 into the plasmidpUC19 to produce the plasmid pBG39, and conferred Bialaphos resistanceto E. coli, W3110, C600 and JM83.

Using an in vitro coupled transcription-translation system (ref. 5) fromS. lividans extracts, the 1,65 Kb PstI-BamHI fragment in pBG39 was shownto direct the synthesis of a 22 Kd protein. In the following, this 1,65Kb insert includes a fragment coding for a 22 Kd protein and will becalled “sfr” gene.

Fine Mapping and Sequencing of the Gene

A 625 bp Sau3A fragment was subcloned from pBG39 into pUC19 and stillconferred Bialaphos resistance to a E. coli W3110 host. The resultingclones were pBG93 and pBG94, according to the orientation.

The orientation of the gene in the Sau3A fragment was indicated byexperiments which have shown that Bialaphos resistance could be inducedwith IPTG from the pUC19 lac promoter in pBG93. In the presence of IPTG(0.5 mM) the resistance of pBG93/W3110 increased from 5 to 50 μg/ml on aM9 medium containing Bialaphos. The W3110 host devoid of pBG93, did notgrow on M9 medium containing 5 μg/ml Bialaphos. These experimentsdemonstrated that the Sau3A fragment could be subcloned without loss ofactivity. They also provided for the proper orientation as shown in theFIG. 2, enclosed thereafter. The protein encoded by these clones wasdetected by using coupled transcription-translation systems derived fromextracts of S. lividans (ref. 7). Depending on the orientation of theSau3A fragment, translation products of different sizes were observed;22 Kd for pBG94 and ±28 Kd for pBG93. This indicated that the Sau3Afragment did not contain the entire resistance gene and that a fusionprotein was formed which included a polypeptide sequence resulting fromthe translation of a pUC19 sequence.

In order to obtain large amounts of the protein, a 1.7 Kb PstI fragmentfrom pBG1 was cloned into the high copy number Streptomycete repliconpIJ385. The obtained plasmid, pBG20, was used to transform S.hygroscopicus. Transformants which contained this plasmid had more than5 times as much PPT acetylating activity and also had increased amountsof a 22 kd protein on sodium dodecylsulfate gels (SDS gels).Furthermore, both the acetyl transferase and the 22 kd protein appearedwhen the production of Bialaphos begun. The correlation of the in vitrodata, kinetics of synthesis, and amplified expression associated withpBG20 transformants strongly implied that this 22 Kd band was the geneproduct.

The complete nucleotide sequence of the 625 bp Sau3A fragment wasdetermined as well as of flanking sequences. Computer analysis revealedthe presence of an open reading frame over the entire length of theSau3A fragment.

Characterization of the sfr Gene Product

A series of experiments were performed to determine that the openreading frame of the “sfr” gene indeed encoded the Bialaphos resistanceenzyme. To determine the 5′ end of the resistance gene, the NH₂-terminalsequence of the enzyme was determined. As concerns more particularly thetechnique used to determine the said sequence, reference is made to thetechnique developed by J. VANDEKERCKHOVE, Eur. J. Bioc. 152, p. 9-19,1985, and to French patent applications No. 85 14579 filed on Oct. 1,1985 and No. 85 13046 filed on September 2, 1985, all of which areincorporated herein by reference.

This technique allows the immobilization on glass fibre sheets coatedwith the polyquaternary amine commercially available under theregistered trademark POLYBRENE of proteins and of nucleic acidspreviously separated on a sodium dodecylsulfate containingpolyacrylamide gel. The transfer is carried out essentially as for theprotein blotting on nitrocellulose membranes (ref. 8). This allows thedetermination of amino-acid composition and partial sequence of theimmobilized proteins. The portion of the sheet carrying the immobilized22 kd protein produced by S. hygroscopicus pBG20 was cut out and thedisc was mounted in the reaction chambre of a gas-phase sequenator tosubject the glass-fibre bound 22 Kd protein to the Edman degradationprocedure. The following amino-acid sequence was obtained SEQ ID NO:4):

Pro-Glu-Arg-Arg-Pro-Ala-Asp-Ile-Arg-Arg

This sequence matched an amino-acid sequence which was deduced from theopen reading frame of the 625 bp Sau3A fragment. It corresponded to thestretch from codon 3 to codon 12.

Thus, the NH₂-terminus of the 22 Kd protein was upstream of thissequence. It was determined that translation of the actual protein waslikely to be initiated 2 amino-acids earlier at a GTG initiation codon.GTG is often used as initiator codon in Streptomyces and translated asmethionine. The protein translated from the GTG initiation codon wouldbe 183 amino-acids long and would have a molecular weight of 20 550.This was in good agreement with the observed approximate molecularweight of 22 000.

Furthermore, the termination codon, TGA, was located just downstream ofthe Sau3A site. Cloning of the 625 bp Sau3A fragment in a BamHI sitedigested pUC19 did not result in the reconstruction of the terminationcodon. This explained the fusion proteins which were observed in the invitro transcription-translation analysis.

Mechanism of PPT-Resistance

Having defined a first phenotype and some of the physicalcharacteristics of the resistance gene and its gene product, a series ofexperiments was then carried out to understand the mechanism by which itconfers resistance. As described hereabove, PPT is the portion ofBialaphos which inhibits glutamine synthetase (GS) and that N-acetyl PPTis not an inhibitor. Using a standard assay (ref. 9), S. hygroscopicusATCC 21 705 derivates were shown to contain a PPT acetyl transferasewhich was not found in S. lividans. The activity does not acetylate theBialaphos tripeptide. S. lividans carrying the resistance gene cloned inpBG20 or pBG16 (a plasmid containing the 625 bp Sau3A fragment clonedinto another streptomycete vector, pIJ680) also contained the activitywhich could acetylate PPT but not Bialaphos. The PPT derived reactionproduct produced by extracts of pBG20/S. lividans was isolated in orderto confirm that it was indeed acetyl-PPT. Analysis by mass spectroscopyshowed that the molecular weight had increased relative to PPT by theequivalent of one acetyl group. It was thus concluded that the 625 bpSau3A fragment contained sequences which code for PPT acetyltransferase.

The experimental conditions and reagents used in the techniquesdisclosed hereabove were as follows:

Preparation and Composition of the Mediums and Buffers Above Used

1° P medium: 10.3 g of sucrose, 0.025 g of K₂SO₄, 0.203 g of MgCl₂.6H₂Oand 0.2 ml of a trace element solution are dissolved in 80 ml ofdistilled water and autoclaved. Then in order, 1 ml of KH₂PO₄ (0.5%), 10ml of CaCl₂, 2H₂O (3.68%), and 10 ml of TES buffer (0.25 M), pH: 7.2)are added. Trace element solution (per liter): ZnCl₂, 40 mg; FeCl₃.6H₂O,200 mg; CuCl₂.2H₂O, 10 mg; MnCl₂.4H₂O, 10 mg; Na₂B₄O₇.10H₂O, 10 mg;(NH₄)₆Mo₇O₂₄.4H₂O, 10 mg.

2° R2YE: 10.3 g of sucrose, 0.025 g of K₂SO₄, 1.012 g of MgCl₂.6H₂O, 1 gof glucose, 0.01 g of Difco 25 casamino acids, and 2.2 g of Difco agarare dissolved in 80 ml distilled water and autoclaved. 0.2 ml of traceelement solution, 1 ml of KH₂PO₄ (0.5%), 8.02 ml of CaCl₂.2H₂O (3.68%),1.5 ml of L-proline (20%), 10 ml of TES buffer (0.25 M) (pH: 7.2), 0.5ml of (1 M) NaOH, 5 ml of yeast extract (10%) are sequentially added.

3° TE: 10 mM TRIS HCl, 1 mM EDTA, pH 8.0.

4° YEME: Difco yeast extract (0.3%), Difco peptone (0.5%), oxoid maltextract (0.3%), glucose (1%).

Transformation of S. lividans Protoplasts

-   1. A culture composed of 25 ml YEME, 34% sucrose, 0.005 M MgCl₂,    0.5% glycine, in a 250 ml baffled flask, is centrifuged during 30 to    36 hours.-   2. The pellet is suspended in 10.3% sucrose and centrifuged. This    washing is repeated once.-   3. The mycelium is suspended in 4 ml lysozyme solution (1 mg/ml in P    medium with CaCl₂ and MgCl₂ concentrations reduced to 0.0025 M) and    incubated at 30° C. for 15 to 60 minutes.-   4. The solution is mixed by pipetting three times in a 5 ml pipette    and incubated for further 15 minutes.-   5. P medium (5 ml) is added and mixed by pipetting as in step 4.-   6. The solution is filtered through cotton wool and protoplasts are    gently sedimented in a bench centrifuge at 800×G during 7 minutes.-   7. Protoplasts are suspended in 4 ml P medium and centrifuged again.-   8. Step 7 is repeated and protoplasts are suspended in the drop of P    medium left after pouring off the supernatant (for transformation).-   9. DNA is added in less than 20 μl TE.-   10. 0.5 ml PEG 1 000 solution (2.5 g PEG dissolved in 7.5 ml of 2.5%    sucrose, 0.0014 K₂SO₄, 0.1 M CaCl₂, 0.05 M TRIS-maleic acid, pH 8.0,    plus trace elements) is immediately added and pipetted once to mix    the components.-   11. After 60 seconds, 5 ml of P medium are added and the protoplasts    are sedimented by gentle centrifugation.-   12. The pellet is suspended in P medium (1 ml).-   13. 0.1 ml is plated out on R2YE plates (for transformation dry    plates to 85% of their fresh weigh e.g. in a laminar flow cabinet).-   14. Incubation at 30° C.

A—Construction of a “sfr” Gene Cassette

A “sfr” gene cassette was constructed to allow subsequent cloning inplant expression vectors.

Isolation of a FokI-BglII fragment from the plasmid pBG39 containing a“sfr” gene fragment led to the loss of the first codons, including theinitiation codon, and of the last codons, including the stop codon.

This fragment of the “sfr” gene could be reconstructed in vitro withsynthetic oligonucleotides which encode appropriate amino-acids.

The complementary synthetic oligonucleotides were (SEQ ID NOS:5-6)5′-CATGAGCCCAGAAC and 3′-TCGGGTCTTGCTGC.

By using such synthetic oligonucleotides, the 5′ end of the “sfr” genecould be reformed and the GTG initiation codon substituted for a codonwell translated by plant cells, particularly an ATG codon.

The DNA fragment containing the oligonucleotides linked to the “sfr”gene was then inserted into an appropriate plasmid, which contained adetermined nucleotide sequence thereafter designated by an “adapter”fragment.

This adapter fragment comprised:

a TGA termination codon which enabled the last codons of the “sfr” geneto be reformed;

appropriate restriction sites which enabled the insertion of thefragment of the nucleotide sequence comprising the “sfr” gene partiallyreformed with the synthetic oligonucleotides; this insertion resulted inthe reconstruction of an intact “sfr” gene;

appropriate restriction sites for the isolation of the entire “sfr”gene.

The “sfr” gene was then inserted into another plasmid, which contained asuitable plant promoter sequence. The plant promoter sequence consistedof the cauliflower mosaic virus promoter sequence (p35S). Of course theinvention is not limited to the use of this particular promoter. Othersequences could be chosen as promoters suitable in plants, for examplethe TR 1′-2′ promoter region and the promoter fragment of a Rubiscosmall subunit gene from Argbidopsis thaliana hereafter described.

1° Construction of the Plasmid pLK56.2 (FIG. 3)

The construction of plasmid pLK56.2 aimed at obtaining a suitableadaptor including the following sequence of restriction sites: SmaI,BamHI, NcoI, KpnI, BglII, MluI, BamHI, HindIII and XbaI.

The starting plasmids used for this construction, pLK56, pJB64 and pLK33were those disclosed by BOTTERMAN (ref. 11).

The DNA fragments hereafter described were isolated and separated fromlow melting point agarose (LGA).

The plasmid pLK56 was cleaved by the enzymes BamHI and NdeI. A NcoI-NdeIfragment (referred to in the drawings by arc “a” in broken line)obtained from plasmid pJB64 was substituted in pLK56 for the BamHI-NdeIfragment shown at “b”. Ligation was possible after filling in the BamHIand NcoI protruding ends with the DNA polymerase I of E. coli (Klenow'sfragment).

Particularly recircularization took place by means of a T4 DNA ligase. Anew plasmid pLK56.3 was obtained.

This plasmid was cleaved by the enzymes XbaI and PstI.

The BamHI-PstI fragment of pLK33 (c) (on FIG. 3) was substituted for theXbaI-PstI fragment (d) of pLK56.3, after repairing of their respectiveends by Klenow's fragment.

After recircularization by means of the T4 DNA ligase, the obtainedplasmid pLK56.2 contained a nucleotide sequence which comprised thenecessary restriction sites for the subsequent insertion of the “sfr”gene.

2° Construction of the Plasmid pGSH150 (FIG. 4A)

Parallel with the last discussed construction, there was produced aplasmid containing a promoter sequence recognized by the polymerases ofplant cells and including suitable restriction sites, downstream of saidpromoter sequence in the direction of transcription, which restrictionsites are then intented to enable the accomodation of the “sfr” genethen obtainable from pLK56.2, under the control of said plant promoter.

Plasmid pGV825 is described in DEBLAERE et al. (ref. 10). Plasmid pJB63is from BOTTERMAN (ref. 11).

pGV825 was linearized with PvuII and recircularized by the T4 DNAligase, resulting in the deletion of an internal PvuII fragment shown at(e), (plasmid pGV956).

pGV956 was then cleaved by BamHI and BglII.

The BamHI-HindIII fragment (f) obtained from pJB63 was dephosphorylatedwith calf intestine phosphatase (CIP) and substituted for theBamHI-BglII fragment of pGV956.

Plasmid pGV1500 was obtained after recircularization by means of T4 DNAligase.

An EcoRI-HindIII fragment obtained from plasmid pGSH50 was purified. Thelatter plasmid carried the dual TR 1′-2′ promoter fragment described inVELTEN et al., (ref. 13). This fragment was inserted in pGV1500,digested with HpaI and HindIII and yielded pGSH150.

This plasmid contains the promoter fragment in front of the 3′ end ofthe T-DNA transcript 7 and a BamHI and ClaI sites for cloning.

3° Construction of the Plasmid pGSJ260 (FIG. 4B)

CP3 is a plasmid derived from pBR322 and which contains the 35S promoterregion of cauliflower mosaic virus within a BamHI fragment.

pGSH150 was cut by BamHI and BglII.

The BamHI-BglII fragment (h) of CP3, which contained the nucleotidesequence of p35S promoter, was substituted for the BamHI-BglII fragment(i) in pGSH150 to form plasmid pGSJ250. pGSJ250 was then opened at itsBglII restriction site.

A BamHI fragment obtained from mGV2 (ref. 12) was inserted in pGSJ250 atthe BglII site to form plasmid pGSJ260.

However prior to inserting the “sfr” gene obtainable from pLK56.2 intoplasmid pGSJ260, it was still desirable to further modify the first inorder to permit insertion in a more practical manner. Thus pLK56.2 wasfurther modified as discussed below to yield pGSR1.

Starting from plasmid pGSJ260, two plasmid constructions for subsequenttransformations of plant cells were made:

a first plasmid permitting the expression of the “sfr” gene in thecytoplasm of plant cells, and

a second plasmid so modified as to achieve transport of theBialaphos-resistance enzymes to the chloroplasts of plant cells.

First Case: Plasmid Enabling the Expression of the “sfr” Gene in theCytoplasm of Plant Cells

Cloning of the sfr Gene Cassette in a Plant Expression Vector (pGSR2)(FIG. 5)

On FIG. 5A (SEQ ID NO:13), the nucleotide sequence of the adapter ofpLK56.2 is shown. In particular, the locations of BamHI, NcoI, BglIIrestriction sites are shown.

This adapter fragment was cleaved by the enzymes NcoI and BglII.

FIG. 5B (SEQ ID NO:14) shows the FokI-BglII fragment (j) obtained frompBG39. The locations of these two restriction sites are shown on FIG. 2.

Using synthetic oligonucleotides, the first codons of the “sfr” genewere reformed, particularly the 5′ end of the gene in which a ATGinitiation codon was substituted for the initial GTG codon.

This FokI-BglII fragment completed with the synthetic oligonucleotideswas then substituted in pLK56.2 for the NcoI-BglII fragment of theadapter. The 3′ end of the gene was thus reformed too, afterrecircularization with T4 DNA ligased. The plasmid obtained, pGSR1, thuscontained the entire “sfr” gene inserted in its adapter.

The plasmid pGSJ260 was then opened by BamHI (FIG. 5C) and the BamHIfragment obtained from pGSR1, which contained the entire “sfr” gene, wasinserted into pGSJ260.

The obtained plasmid, pGSR2 (see FIG. 6A) contained a pBR322 replicon, abacterial streptomycin resistance gene (SDM-SP-AD-transferase) and anengineered T-DNA consisting of:

the border fragments of the T-DNA;

a chimeric kanamycin gene which provided a dominant selectable marker inplant cells; and

a chimeric “sfr” gene.

The chimeric “sfr” gene consisting of:

the promoter region of the cauliflower mosaic virus (p35S);

the “sfr” gene cassette as described in FIG. 5;

the 3′ untranslated region, including the polyadenylation signal ofT-DNA transcript 7.

pGSR2 was introduced into Agrobacterium tumefaciens recipient C58ClRif®(pGV2260) according to the procedure described by DEBLAERE et al. (ref.10).

This strain was used to introduce the chimeric “sfr” gene in N. tabacumSR₁ plants.

Two variant plasmids deriving from pGSR2, namely pGSFR280 and pGSFR281,have been constructed. They differ in the untranslated sequencefollowing the transcription initiation site. In pGSR2, this fragmentconsists of the following sequence (SEQ ID NO:7):

GAGGACACGCTGAAATCACCAGTCTCGGATCC ATG;

while it consists of (SEQ ID NO:8):

GAGGACACGCTGAAATCACCAGTCTCTC- TACAAATCGATCCATG

in pGSR280 and of (SEQ ID NO:9)

GAGGACACGCTGAAATCACCAGTCTCTC- TACAAATCGATG

in pGSFR281, with an ATG codon being the initiation codon of the “sfr”gene. The “sfr” gene is also fused to the TR1′-2′ promoter in theplasmid pGSH150 (FIG. 4A) yielding pGSFR160 and pGSFR161 (FIG. 6B).These plasmids contain slight differences in the pTR2 “sfr” geneconfiguration: the “sfr” gene is correctly fused to the endogenous gene2′ ATG in pGSFR161 (for sequences see ref. 13), whereas 4 extra basepairs (ATCC) are present just ahead of the ATG codon in pGSFR160.Otherwise, plasmids p65FR161 and p65FR160 are completely identical.

All plasmids are introduced in Agrobacterium by cointegration in theacceptor plamids pGV2260 yielding the respective plasmids pGSFR1280,pGSFR1281, pGSFR1160 and pGSFR1161.

Second Case: Construction of a Plasmid Containing the “sfr” GeneDownstream of a DNA Sequence Encoding a Transit Peptide and Suitable forAchieving Subsequent Translocation of the “sfr” Gene Expression Productinto Plant-Cell-Chloroplasts

In another set of experiments, the nucleotide sequence which containedthe “sfr” gene was fused to a DNA sequence encoding a transit peptide soas to enable its transport into chloroplasts.

A fragment of the “sfr” gene was isolated from the adapter fragmentabove described and fused to a transit peptide. With syntheticoligonucleotides, the entire “sfr” gene was reconstructed and fused to atransit peptide.

The plasmid (plasmid pATS3 mentioned below) which contained thenucleotide sequence encoding the transit peptide comprised also thepromoter sequence thereof.

Construction of the Plasmid pGSR4 which Contains the “sfr” Gene Fused toa DNA Sequence Encoding Transit Peptide (FIG. 7)

Plasmid pLK57 is from BOTTERMAN, (ref. 11). Plasmid pATS3 is a pUC19clone which contains a 2 Kb EcoRI genomic DNA fragment from Arabidopsisthaliana comprising the promoter region and the transit peptidenucleotide sequence of the gene, the expression thereof is the smallsubunit of ribulose biphosphate carboxylase (ssu). The A. thaliana smallsubunit was isolated as a 1 500 bp EcoRI-SphI fragment. The SphIcleavage site exactly occurs at the site where the coding region of themature ssu protein starts.

Plasmids pLK57 and pATS3 were opened with EcoRI and SphI. Afterrecircularization by means of the T4 DNA ligase, a recombinant plasmidpLKAB1 containing the sequence encoding the transit peptide (Tp) and itspromoter region (Pssu) was obtained.

In order to correctly fuse the “sfr” gene at the cleavage site of thesignal peptide, the N-terminal gene sequence was first modified. Sinceit was observed that N-terminal gene fusions with the “sfr” gene retaintheir enzymatic activity, the second codon (AGC) was modified to a GAC,yielding an NcoI site overlapping with the ATG initiator site. A newplasmid, pGSSFR2 was obtained. It only differs from pGSR1 (FIG. 5B), bythat mutation. The NcoI-BamHI fragment obtained from pGSFR2 was fused atthe SphI end of the transit peptide sequence. In parallel, the “sfr”gene fragment was fused correctly to the ATG initiator of the ssu gene(not shown in figures).

Introduction of the “sfr” Gene into a Different Plant Species

The Bialaphos-resistance induced in plants by the expression of chimericgenes, when the latter have been transformed with appropriate vectorscontaining said chimeric genes, has been demonstrated as follows. Therecombinant plasmids containing the “sfr” gene were introducedseparately by mobilization into Agrobacterium strain C58C₁ Rif®(pGV2260) according to the procedure described by DEBLAERE and al.,Nucl. Acid. Res., 13, p. 1 477, 1985. Recombinant strains containinghybrid Ti plasmides were formed. These strains were used to infect andtransform leaf discs of different plant species, according to a methodessentially as described by HORSH and al., 1985, Science, vol. 227.Transformation procedure of these different plant species given by wayof example, is described thereafter.

-   1. Leaf Disc Transformation of Nicotiana tabacum

Used Media are described thereafter:

A₁ MS salt/2 +1% sucrose 0.8% agar pH 5.7 A₁₀ B5-medium +250 mg/l NH₄NO₃750 mg/l CaCl₂ 2H₂O 0.5 g/l 2-(N-Morpholino)ethane- sulfonic acid (MES)pH 5.7 30 g/l sucrose A₁₁ B5-medium +250 mg/l NH₄NO₃ 0.5 g/l MES pH 5.72% glucose 0.8% agar 40 mg/l adenine +1 mg/l 6-Benzylaminopurine (BAP)0.1 mg/l Indole-3-acetic acid (IAA) 500 mg/l Claforan A₁₂ B5-medium +250mg/l NH₄NO₃ 0.5 g/l MES pH 5.7 2% glucose 0.8% agar 40 mg/l adenine +1mg/l BAP 200 mg/l claforan A₁₃ MS-salt/2 +3% sucrose 0.5 MES g/l pH 5.70.7% agar 200 mg/l claforan Bacterial medium = min A: (Miller 1972) 60mM K₂HPO₄, 3H₂O, 33 mM KH₂PO₄; 7.5 mM (NH₄)₂ SO4 1.7M trinatriumcitrat;1 mM MgSO₄; 2 g/l glucose; 50 mg/l vitamine B1

Plant Material:

Nicotiana tabacum cv. Petit Havana SR1

Plants are used 6 to 8 weeks after subculture on medium A₁

Infection:

midribs and edges are removed from leaves.

Remaining parts are cut into segments of about 0.25 cm² and are placedin the infection medium A₁₀ (about 12 segments in a 9 cm Petri dishcontaining 10 ml A₁₀).

Segments are then infected with 25 μl per Petri dish of a late logculture of the Agrobacterium strain grown in min A medium.

Petri dish are incubated for 2 to 3 days at low light intensity.

After 2 to 3 days medium is removed and replaced by 20 ml of medium A₁₀containing 500 mg/l clarofan.

Selection and Shoot Induction

The leaf discs are placed on medium A₁₁ containing a selective agent:

100 mg/l kanamycin and

10 to 100 mg/l phosphinotricin.

Leaf discs are transferred to fresh medium weekly.

After 3 to 4 weeks regenating calli arise. They are separated and placedon medium A₁₂ with the same concentration of selective agent as used forthe selection.

Rooting

After 2 to 3 weeks the calli are covered with shoots, which can beisolated and transferred to rooting medium A₁₃ (without selection).

Rooting takes 1 to 2 weeks.

After a few more weeks, these plants are propagated on medium A₁.

-   2. Tuber Disc Infection of Solanum tuberosum (Potato)

Used media are described thereafter:

C₁ B5-medium +250 mg/l NH₄NO₃ 300 mg/l (CaCH₂PO₄)₂ 0.5 g/l MES pH 5.70.5 g/l polyvinylpyrrolidone (PVP) 40 g/l mannitol (=0.22M) 0.8% agar 40mg/l adenine C₂ B5-medium +250 mg/l NH₄NO₃ 400 mg/l glutamine 0.5 g/lMES pH 5.7 0.5 g/l PVP 40 g/l mannitol 40 mg/l adenine 0.8% agar +0.5mg/l transzeatine 0.1 mg/l IAA 500 mg/l clarofan C₅ MS salt/2 +3%sucrose 0.7% agar pH 5.7 C₇ B5-medium +250 mg/l NH₄NO₃ 400 mg/lglutamine 0.5 g/l MES pH 5.7 0.5 g/l PVP 20 g/l mannitol 20 g/l glucose40 mg/l adenine 0.6% agarose +0.5 mg/l transzeatine 0.1 mg/l IAA 500mg/l clarofan C₈ B5-medium +250 mg/l NH₄NO₃ 400 mg/l glutamine 0.5 g/lMES pH 5.7 0.5 g/l PVP 20 g/l mannitol 20 g/l glucose 40 mg/l adenine0.6% agarose +200 mg/l clarofan 1 mg/l transzeatine C₉ B5-medium +250mg/l NH₄NO₃ 400 mg/l glutamine 0.5 g/l MES pH 5.7 0.5 g/l PVP 20 g/lmannitol 20 g/l glucose 40 mg/l adenine 0.6% agarose +1 mg/ltranszeatine 0.01 mg/l Gibberellic acid A₃ (GA₃) 100 mg/l clarofan C11MS salt/2 +6% sucrose 0.7% agar Bacterial medium = min (Miller 1972 60mM K₂HPO₄•3H₂O; A: 33 mM KH₂PO₄; 7.5 mM (NH₄)₂SO₄; 1.7 trinatriumcitrat;1 mM MgSO₄; 2 g/l glucose; 50 mg/l vitamine B1.

Plant Material

Tubers of Solanum tuberosum c.v Berolina

c.v Désirée

Infection

Potatoes are peeled and washed with water.

Then they are washed with concentrated commercial bleach for 20 minutes,and

rinsed 3 to 5 times with sterile water.

The outer layer is removed (1 to 1.5 cm)

The central part is cut into discs of about 1 cm² and 2 to 3 mm thick.

Discs are placed on medium C₁ (4 pieces in a 9 cm Petri dish).

10 μl of a late log culture of an Agrobacterium strain grown in min Amedium is applied on each disc.

Discs are incubated for 2 days at low light intensity.

Selection and Shoot Induction

Discs are dried on a filter paper and transferred to medium C₂ with 100mg/l kanamycin.

After one month small calli are removed from the discs and transferredto medium C₇ containing 50 mg/l kanamycin.

After a few more weeks, the calli are transferred to medium C₈containing 50 mg/l kanamycin.

If little shoots start to develop, the calli are transferred toelongation medium C₉ containing 50 mg/l Kanamycin.

Rooting

Elongated shoots are separated and transferred to rooting medium C₁₁.

Rooted shoots are propagated on medium C₅.

-   3. Leaf Disc Infection of Lycopersicum esculentum (Tomato)

Used media are described thereafter

A₁ MS salt/2 +1% sucrose 0.8% agar pH 5.7 B₁ B5-medium +250 mg/l NH₄NO₃0.5 g/l MES pH 5.7 0.5 g/l PVP 300 mg/l Ca (H₂PO₄)₂ 2% glucose 40 mg/ladenine 40 g/l mannitol B₂ B5-medium +250 mg/l NH₄NO₃ 0.5 g/l MES pH 5.70.5 g/l PVP 400 mg/l glutamine 2% glucose 0.6% agarose 40 mg/l adenine40 g/l mannitol +0.5 mg/l transzeatine 0.01 mg/l IAA 500 mg/l claforanB₃ B5-medium +250 mg/l NH₄NO₃ 0.5 g/l MES pH 5.7 0.5 g/l PVP 400 mg/lglutamine 2% glucose 0.6% agarose 40 mg/l adenine 30 g/l mannitol +0.5mg/l transzeatine 0.01 mg/l IAA 500 mg/l clarofan B₄ B5-medium +250 mg/lNH₄NO₃ 0.5 g/l MES pH 5.7 0.5 g/l PVP 400 mg/l glutamine 2% glucose 0.6%agarose 40 mg/l adenine 20 g/l mannitol +0.5 mg/l transzeatine 0.01 mg/lIAA 500 mg/l clarofan B₅ B5-medium +250 mg/l NH₄NO₃ 0.5 g/l MES pH 5.70.5 g/l PVP 400 mg/l glutamine 2% glucose 0.6% agarose 40 mg/l adenine10 g/l mannitol +0.5 mg/l transzeatine 0.01 mg/l IAA 500 mg/l clarofanB₆ B5-medium +250 mg/l NH₄NO₃ 0.5 g/l MES pH 5.7 0.5 g/l PVP 400 mg/lglutamine 2% glucose 0.6% agarose 40 mg/l adenine +0.5 mg/l transzeatine0.01 mg/l IAA 200 mg/l clarofan B₇ B5-medium +250 mg/l NH₄NO₃ 0.5 g/lMES pH 5.7 0.5 g/l PVP 400 mg/l glutamine 2% glucose 0.6% agarose 40mg/l adenine +1 mg/l transzeatine 200 mg/l clarofan B₈ MS salt/2 +2%sucrose 0.5 g/l MES pH 5.7 0.7% agar B₉ B5-medium +250 mg/l NH₄NO₃ 0.5g/l MES pH 5.7 0.5 g/l PVP 2% glucose 0.6% agarose 40 mg/l adenine +1mg/l transzeatine 0.01 mg/l GA3 Bacterial medium = min (Miller 1972) 60mM A: K₂HPO₄•3H₂O; 33 mM KH₂PO₄; 7.5 mM (NH₄)₂SO4; 1.7Mtrinatriumcitrat; 1 mM MgSO₄; 2 g/l glucose; 50 mg/l vitamin B1

Plant Material

Lycopersicum esculentum cv. Lucullus.

Plants are used 6 weeks after subculture on medium A₁.

Infection

Midrib is removed from the leaves.

Leaves are cut in segments of about 0.25 to 1 cm² (the edges of theleaves are not wounded, so that only maximum 3 sides of the leaf piecesis wounded).

Segments are placed in infection medium B₁ (upside down), about 10segments in a 9 cm Petri dish.

Segments are then infected wiht 20 μl per Petri dish of a late logculture of the Agrobacterium strain grown in min A medium.

Petri dishes incubate for 2 days at low light intensity.

Medium is removed after 2 days and replaced by 20 ml of medium B₁containing 500 mg/l clarofan.

Selection and Shoot Induction

The leaf discs are placed in medium B₂+50 or 100 mg/l kanamycin.

Each 5 days the osmotic pressure of the medium is lowered by decreasingthe mannitol concentration, transfers are done consecutively in mediumB₃, B₄, B₅, and B₆.

After one month calli with meristems are separated from the leaf discsand placed on medium B₇ with 50 or 100 mg/l kanamycin.

Once little shoots have formed, calli are transferred to elongationmedium B₉ with 50 or 100 mg/l kanamycin.

Rooting

Elongated shoots are separated and transferred to medium B₈ for rooting.

Plants are propagated on medium A₁.

Greenhouse Tests for Herbicide Resistance

Material and Method

In this experiment, two herbicides comprising phosphinotricin as activeingredient, are used.

These compounds are those commercially available under the registeredtrademarks BASTA® and MEIJI HERBIACE®.

These products are diluted to 2% with tap water. Spraying is carried outon a square meter area from the four corners. Temperature of thegreenhouse is about 22° C. for tobaccos and tomato, and above 10° C. to15° C. for potato.

Results

Tobacco Spraytest

a) Nicotiana tabacum cv. Petit Havana SR1 plants transformed with thechimeric “sfr” genes as present in pGSFR1161 or pGSFR1281, as well asunstransformed control plants (from 10 cm to 50 cm high) are treatedwith 20 1 BASTA®/ha. Control SR1 plants die after 6 days, whiletransformed plants are fully resistant to 20 1 BASTA®/ha and continuegrowing undistinguishable from untreated plants. No visible damage isdetected, also the treatment is repeated every two weeks. The treatmenthas no effect in subsequent flowering. The recommended dose oF BASTA®herbicide in agriculture is 2.5-7.5 l/ha.

b) A similar experiment is performed using 8 l/ha MEIJI HERBIACE®. Thetransformed plants (the same as above) are fully resistant and continuegrowing undistinguishable from untreated plants. No visible damage isdetectable.

Potato Spraytest

Untransformed and transformed potato plants (Solanum tuberosum cv.Berolina) (20 cm high) with the chimeric “sfr” gene as present inpGSFR1161 or pGSFR1281 are treated with 20 1 BASTA®/ha. Control plantsdie after 6 days while the transformed plants do not show any visibledamage. They grow undistiguishable from untreated plants.

Tomato Spraytest

Untransformed and transformed tomato plants (lycopersium esculentum c.v.luculus) (25 cm high) with the chimeric “sfr” gene as present inpGSFR1161 and pGSFR1281 are treated with 20 1 BASTA®/ha. control plantsdie after six days while transformed plants are fully resistant. They donot show any visible damage and grow undistiguishable from untreatedplants.

Growth Control of Phytopathogenic Fungi with Transformed Plants

In another set of experiments, potato plants expressing chimeric “sfr”genes as present in pGSFR1161 or pGSFR1281 are grown in a greenhousecompartment at 20° C. under high humidity. Plants are innoculated byspraying 1 ml of a suspension of 10⁶ Phytophtora infestans spores perml. Plants grow in growth chambers (20° C., 95% humidity, 4 000 lux)until fungal disease symptoms are visible (one week). One set of theplants are at that moment sprayed with Bialaphos at a dose of 8 l/ha.Two weeks later, untreated plants are completely ingested by the fungus.The growth of the fungus is stopped on the Bialaphos treated plants andno further disease symptoms evolve. The plants are effectively protectedby the fungicide action of Bialaphos.

Transmission of the PPT Resistance through Seeds

Transformed tobacco plants expressing the chimeric “sfr” gene present inpGSFR1161 and pGSFR1281 are brought to flowering in the greenhouse. Theyshow a normal fertility.

About 500 F1 seeds of each plant are sown in soil, F1 designating seedsof the first generation, i.e directly issued from the originallytransformed plants. When seedlings are 2-3 cm high, they are sprayedwith 8 1 BASTA®/ha. 7 days later, healthy and damaged plants can bedistinguished in a ratio of approximately 3 to 1. this shows that PPTresistance is inherited as a dominant marker encoded by a single locus.

10 resistant F1 seedlings are grown to maturity and seeds are harvested.F2 seedlings are grown as described above and tested for PPT-resistanceby spraying BASTA® at a dose of 8 l/ha. Some of the F1 plants produce F2seedlings which are all PPT-resistant showing that these plants arehomozygous for the resistance gene. The 5 invention also concerns plantcells and plants non-essentially-biologically-transformed with a GSinhibitor-inactivating-gene according to the invention.

In a preferred embodiment of the invention, plant cells and plants arenon-biologically-transformed with the “sfr” gene hereabove described.

Such plant cells and plants possess, stably integrated in their genome,a non-variety-specific character which render them able to producedetectable amounts of phosphinotricinacetyl transferase.

This character confers to the transformed plant cells and plants anon-variety-specific enzymatic activity capable of inactivating orneutralizing GS inhibitors like Bialaphos and PPT.

Accordingly, plant cells and plants transformed according to theinvention are rendered resistant against the herbicidal effects ofBialaphos and related compounds.

Since Bialaphos was first described as a fungicide, transformed plantscan also be protected against fungal diseases by spraying with thecompound several times.

In a preferred embodiment, Bialaphos or related compounds is appliedseveral times, particularly at time intervals of about 20 to 100 days.

The invention also concerns a new process for selectively protecting aplant species against fungal diseases and selectively destroying weedsin a field comprising the steps of treating a field with an herbicide,wherein the plant species contain in their genome a DNA fragmentencoding a protein having an enzymatic activity capable of neutralizingor inactivating GS inhibitors and wherein the used herbicide comprisesas active ingredient a GS inhibitor.

It comes without saying that the process according to the invention canbe employed with the same efficiency, either to only destroy weeds in afield, if plants are not infected with fungi, either to only stop thedevelopment of fungi if the latter appears after destruction of weeds.

In a preferred embodiment of the process according to the invention,plant species are transformed with a DNA fragment comprising the “sfr”gene as described hereabove, and the used herbicide is PPT or a relatedcompound.

Accordingly, a solution of PPT or related compound is applied over thefield, for example by spraying, several times after emergence of theplant species to be cultivated, until early and late germinating weedsare destroyed.

It is quite evident that before emergence of plant species to becultivated, the field can be treated with an herbicidal composition todestroy weeds.

On the same hand, fields can be treated even before the plant species tobe cultivated are sowed.

Before emergence of the desired plant species, fields can be treatedwith any available herbicide, including Bialaphos-type herbicides.

After emergence of the desired plant species, Bialaphos or relatedcompound is applied several times.

In a preferred embodiment, the herbicide is applied at time intervals ofabout from 20 to 100 days.

Since plants to be cultivated are transformed in such a way as to resistto the herbicidal effects of Bialaphos-type herbicides, fields can betreated even after emergence of the cultivated plants.

This is particularly useful to totally destroy early and lategerminating weeds, without any effect on the plants to be produced.

Preferably, Bialaphos or related compoud is applied at a dose rangingfrom about 0.4 to about 1.6 kg/ha, and diluted in a liquid carrier at aconcentration such as to enable its application to the field at a rateranging from about 2 to about 8 l/ha.

There follows examples, given by way of illustration, of someembodiments of the process with different plant species.

Sugarbeets

The North European sugarbeet is planted from March 15 up to April 15,depending upon the weather condition and more precisely on theprecipitation and average temperature. the weeds problems are more orless the same in each country and can cause difficulties until the cropcloses its canopy around mid-July.

Weed problems can be separated in three situations:

-   -   early germination of the grassy weeds,    -   early germinating broadleaved weeds,    -   late germinating broadleaved weeds.

Up to now, pre-emergence herbicides have been succesfully used. Suchcompounds are for example those commercially available under theregistered trademarks: PYRAMIN®, GOLTIX® and VENZAR®. However, thesusceptibility to dry weather conditions of these products as well asthe lack of residual activity to control late germinating weeds have ledthe farmer to use post-emergence products in addition to pre-emergenceones.

Table (I) thereafter indicates the active ingredients contained in theherbicidal compositions cited in the following examples.

TABLE I Commercial Name Active Ingredient Formulation AVADEX^(R)Diallate EC 400 g/l AVADEX BW^(R) Triallate EC 400 g/l GOLTIX^(R)Metamitron WP 70% RONEET^(R) Cycloate EC 718 g/l TRAMAT^(R) EthofumerateEC 200 g/l FERVINAL^(R) Alloxydime-sodium SP 75% BASTA^(R)Phosphinotricin 200 g/l PYRAMIN FL^(R) Chloridazon SC 430 g/l

According to the invention, post-emergence herbicides consist ofBialaphos or related compounds, which offer a good level of growthcontrol of annual grasses (Bromus, Avena spp., Alopecurus, POA) andbroadleaves (Galium, Polygonum, Senecio, Solanum, Mercurialis).

Post-emergence herbicides can be applied at different moments of thegrowth of sugarbeet; at a cotyledon level, two-leave level or at afour-leave level.

Table (II) thereafter represents possible systems of field-treatment,given by way of example.

In those examples, the post-emergence herbicide of the class ofBialaphos used is BASTA®, in combination with different pre-emergenceherbicides. Concentrations are indicated in l/ha or kg/ha.

TABLE II POSSIBLE WEEDCONTROL SYSTEMS IN SUGARBEETS, BASED ON THE USE OFBASTA^(R), PROVIDING BEETS ARE MADE RESISTANT AGAINST THE LATTERCHEMICAL (in lt or kg/ha). Pre-sowing Pre-emergence CotyledonsTwo-leaves Four leaves 1. AVADEX^(R) — BASTA^(R) BASTA^(R)/tramat — 3.5lt 3 lt 3 lt 1.5 lt — 2. AVADEX^(R) GOLTIX^(R) — — — 3.5 lt 4 kg 3RONEET^(R) GOLTIX^(R) — — — 4 lt 5 kg 4. RONEET^(R) GOLTIX^(R) —BASTA^(R) — 4 lt 2.5 kg 3 lt 5. TRAMAT^(R) — — BASTA^(R)BASTA^(R)/GOLTIX^(R) 5 lt 3 lt 2 lt 2 kg 6. — GOLTIX^(R) — BASTA^(R) —2.5 kg 3 lt 7. — — BASTA^(R)/tramat — BASTA^(R)/GOLTIX^(R) 3 lt 1.7 lt 3lt 2 kg 8. PYRAMIN^(R) — BASTA^(R) Venzar — 6 lt 3 lt 1 kg 9. — —BASTA^(R) BASTA^(R)/GOLTIX^(R) — 3 lt 3 lt 2 kg 10. DIALLATE^(R)PYRAMIN^(R) BASTA^(R)/Metamitron — 3.5 lt 6 lt 3 lt 1 kg

Potatoes

Potatoes are grown in Europe on about 8.10⁶ Ha. The major products usedfor weed control are Linuron/mono-linuron or the compound commerciallyavailable under the denomination METRABUZIN

These products perform well against most weedspecies.

However, weeds such as Galium and Solanum plus late germinatingChenopodium and Polygonum are not always effectively controlled, whilecontrol of the annual grasses is also sometime erratic.

Once again, late germinating broadleaved weeds are only controllable bypost-emergence applications of herbicides such as BASTA®.

Table (III) thereafter represents some examples given by way of exampleof field-treatment in the case of potatoes.

TABLE III Weeds control systems in potatoes, based on the use ofBAST^(R), providing potatoes are rendered resistant to BASTA^(R).Linuron + monolinuron (375 g + 375 g/ha) prior to emergence BASTA^(R)3-4 lt/ha after emergence (5-15 cm) BASTA^(R)/fluazifop-butyl 3-4lt/ha + 2 lt/ha after emergence (5-15 cm) Linuron WP 50% (AFALON^(R))Monolinuron WP 47.5% (ARESSIN^(R)) fluazifop-butyl EL 250 g/l(FUSILADE^(R))

The strains PGSJ260 and pBG39 used hereabove have been deposited on Dec.12, 1985, at the “German Collection of Micro-organisms” (DEUTSCHESAMMLUNG VON MIKROORGANISMEN) at Gottingen, Germany. They received thedeposition numbers DSM 3 606 and DSM 3 607 respectively.

Further embodiments of the invention are described hereafter withreference to the figures in which:

FIG. 8 shows the restriction map of a plasmid pJS1 containing anotherBialaphos-resistance-gene;

FIG. 9 (SEQ ID NO:10) shows the nucleotide sequence of the “sfrsv” genecontaining the resistance gene;

FIG. 10 (SEQ ID NO:18) shows the amino acid homology of “sfrsv” gene and“sfr” gene,

FIG. 11 (SEQ ID NOS:19-21) shows the construction of a plasmid, given byway of example, which contains the “sfrsv” gene and suitable for thetransformation of plant cells.

Another Bialaphos-resistance-gene has been isolated form anotherBialaphos-producing-strains, i.e. streptomyces viridochromogenes. Thissecond resistance-gene is thereafter designated by “sfrsv” gene.

This second preferred DNA fragment according to the invention, for thesubsequent transformation of plant cells, consists of a nucleotidesequence (SEQ ID NO:11) coding for at least part of a polypeptide havingthe following sequence:

V S P E R R P V E I R P A T A A D M A A V C D I V N H Y I E T S T V N PR T E P Q T P Q E W I D D L E R L Q D R Y P W L V A E V E G V V A G I AY A G P W K A R N A Y D W T V E S T V Y V S H R H Q R L G L G S T L Y TH L L K S M E A Q G F K S V V A V I G L P N D P S V R L H E A L G Y T AR G T L R A A G Y K H G G W H D V G F W Q R D F E L P A P P R P V R P VT Q I *which part of said polypeptide is of sufficient length to conferprotection against Bialaphos-“plant-protecting-capability”-, to plantcells, when incorporated genetically and expressed therein. Referencewill also be made hereafter to the “plant-protecting-capability”againstBialaphos of the abovesaid nucleotide sequence.

Meaning of the designation of amino acids by a single letter is giventherafter.

Alanine A Leucine L Arginine R Lysine K Asparagine N Methionine MAspartic Acid D Phenylalanine F Cysteine C Proline P Cystine C Serine SGlycine G Threoriine T Glutamic Acid E Tryptophan W Glutamine Q TyrosineY Histidine H Valine V Isoleucine I

This second preferred DNA fragment consists of the following nucleotidesequence (SEQ ID NO:12):

TAAAGAGGTGCCCGCCACCCGCTTTCGCAGAACACCGAAGGAGACCACAC ↓GTGAGCCCAGAACGACGCCCGGTCGAGATCCGTCCCGCCACCGCCGCCGACATGGCGGCGGTCTGCGACATCGTCAATCACTACATCGAGACGAGCACGGTCAACTTCCGTACGGAGCCGCAGACTCCGCAGGAGTGGATCGACGACCTGGAGCGCCTCCAGGACCGCTACCCCTGGCTCGTCGCCGAGGTGGAGGGCGTCGTCGCCGGCATCGCCTACGCCGGCCCCTGGAAGGCCCGCAACGCCTACGACTGGACCGTCGAGTCGACGGTGTACGTCTCCCACCGGCACCAGCGGCTCGGACTGGGCTCCACCCTCTACACCCACCTGCTGAAGTCCATGGAGGCCCAGGGCTTCAAGAGCGTGGTCGCCGTCATCGGACTGCCCAACGACCCGAGCGTGCGCCTGCACGAGGCGCTCGGATACACCGCGCGCGGGACGCTGCGGGCAGCCGGCTACAAGCACGGGGGCTGGCACGACGTGGGGTTCTGGCAGCGCGACTTCGAGCTGCCGGCCCCGCCCCGCCCCGTCCGGCCCGTCACACAGATCT                                                 ↑ GAGCGGAGAGCGCATGGCor of a part thereof expressing a polypeptide having plant-protectingcapability against Bialaphos;

There follows hereafter the description of experiments carried out forthe isolation of the “sfrsv” resistance gene, the construction ofexpression vectors which contain the resistance gene and which allow thesubsequent transformation of plant cells, in order to render themresistant to GS inhibitors.

Cloning of the Bialaphos-Resistance-“sfrsv” Gene from Streptomycesviridochromogenes

The strain Streptomyces viridochromogenes DSM 40736 (ref 1) was grownand total DNA of this strain was prepared according to standardtechniques. DNA samples were digested respectively with PstI, SmaI andSau3AI in three different reactions and separated on an agarose gel,together with plasmid DNA from pGSR1 (FIG. 5B) digested with BamHI. In aSouthern analysis the DNA was blotted on a nitrocellulose filter andhybridized with the labbeled BamHI fragment from pGSR1 containing the“sfr” gene. In all four lanes of the gel, a restriction fragment wasshowing strong homology with the probe: a PstI fragment of about 3 kb, aSmaI fragment of about 1.2 kb and Sau3AI fragment of 0.5 kb. In order toclone this gene, PstI restriction fragments were directly cloned in theEscherichia coli vector pUC8. 3000 colonies obtained aftertransformation were transferred to nitrocellulose filters, andhybridized with the “sfr” probe. Positive candidates were further testedfor their growth on minimal medium plates containing 300 μg/ml PPT. Onetransformant that grew on PPT-containing-medium was further analysed.The plasmid map and relevant restriction sites of this plasmid pJS1 arerepresented in FIG. 8. The strain MC1061 (pJS1) has been deposited onMar. 6, 1987 at the DEUTSCHE SAMMLUNG VON MIKROORGANISMEN (DSM) underdeposition number DSM 4023. The clone restriction fragment has beensequenced according to the Maxam and Gilbert method and the codingregion of the gene could be identified through homology. The sequence ofthe “sfrsv” gene is represented in FIG. 9 and the homology on thenucleotide and amino acid sequence level with “sfr” gene is shown inFIG. 10.

Expression of the “sfrsv” Gene

A “sfrsv gene cassette” was also constructed to allow subsequent cloningin plant expression vectors. A BanII-BglII fragment containing the“sfrsv” coding region without the initiation codon GTG was isolated fromPJS1. This fragment was ligated in the vector pLK56-2 digested with NcoIand BglII, together with a synthetic oligonucleotide 5′-CATGAGCC-3′,similar with the one described for “sfr” gene and shown in FIG. 5. Theconstruction of pGSR1SV is schematically shown in FIG. 11. Since similarcassettes of both genes are present in respectively pGSR1 and pGSR1SV,previous described constructions for the expression of the “sfr” gene inplants can be repeated.

Enzymatic analysis of crude extracts from E. coli strains carryingplasmid pGSR1SV demonstrated the synthesis of an acetylase which couldacetylate PPT. This was shown by thin layer chromotography of thereaction porducts.

The “sfrsv” gene was then inserted into the plasmid vector pGSJ260 (FIG.4B) under the control of the CaMV 35s promoter, to yield a plasmidpGS2SV, similar to pGSR2 (FIG. 6A) except that the “sfrsv” gene issubstituted for the “sfr” gene.

It is clear that herbicide resistance genes of the above type may beobtained from many other microorganisms that produce PPT or PPTderivatives. Herbicide resistance gene can then be incorporated in plantcells with a view of protecting them against the action of suchGlutamine Synthetase inhibitors. For instance, aBialaphos-resistance-gene is obtained from Kitasotosporia (ref. 15).

Transformed plant cells and plants which contain the “sfrsv” resistancegene can be obtained with plasmid pGSR2SV, using the sameAgrobacterium-mediated-transformation system as hereabove described forthe transformation of different plant species with the “sfr” gene.

Plants are regenerated and tested for their resistance with similarspraying tests as described hereabove. All plants behaved similarly andshow resistance against herbicides consisting of glutamine synthetaseinhibitors.

Finally, the inventors also pertains to the combination of the plantsresistant to an inhibitor of glutamine synthetase as defined above withthe corresponding inhibitor of glutamine synthetase for use in theproduction of cultures of said plants free form weeds.

REFERENCES

-   1. BAYER et al., HELVETICA CHEMICA ACTA, 1972-   2. WAKABAYASHI K. and MATSUNAKA S., Proc. 1982, British Crop    Protection Conference, 439-450-   3. M. MASON et al., PHYTOCHEMISTRY, 1982, vol. 21, No. 4, p.    855-857.-   4. C. J. THOMPSON et al., NATURE, Jul. 31, 1980, vol. 286, No. 5    772, p. 525-527-   5. C. J. THOMPSON et al., JOURNAL OF BACTERIOLOGY, August 1982, p.    678-685-   6. C. J. THOMPSON et al., GENE 20, 1982, p. 51-62-   7. C. J. THOMPSON et al., MOL. GEN. GENET., 1984, 195, p. 39-43-   8. TOWBIN et al., PROC. NATL. ACAD. SCI. USA, 1979, 76, p. 4 350-4    354-   9. METHODS OF ENZYMOLOGY, V.XLIII, p. 737-755-   10. DEBLAERE H. et al., 1985, Nucl. Acid. Res., 13, 1 477-   11. BOTTERMAN J., February 1986, Ph. D. Thesis, State University of    Ghent-   12. DEBLAERE R., February 1986, Ph. D Thesis, Free University of    Brussel, Belgium-   13. VELTEN et al, EMBO J. 1984, vol. 3, No. 12, p. 2 7232-2 730-   14. CHATER et al, Gene cloning in Streptomyces. Curr. Top.    Microbiol. Immunol., 1982, 96, p. 69-75-   15. OMURA et al, J. of Antibiotics, Vol. 37, 8, 939-940, 1984-   16. MURAKAMI et al, Mol. Gen. Genet., 205, 42-50, 1986-   17. MANDERSCHEID and WILD, J. Plant Phys., 123, 135-142, 1986

The invention claimed is:
 1. An isolated DNA encoding a protein havingphosphinothricin acetyltransferase activity, or a variant thereofretaining said activity, said protein comprising the amino acid sequence(SEQ ID No. 1): X   Ser Pro Glu Arg Arg Pro Ala AspIle Arg Arg Ala Thr Glu Ala Asp MET Pro Ala Val Cys Thr Ile Val Asn HisTyr Ile Glu Thr Ser Thr Val Asn Phe Arg Thr Glu Pro Gln Glu Pro Gln GluTrp Thr Asp Asp Leu Val Arg Leu Arg Glu Arg Tyr Pro Trp Leu Val Ala GluVal Asp Gly Glu Val Ala Gly Ile Ala Tyr Ala Gly Pro Trp Lys Ala Arg AsnAla Tyr Asp Trp Thr Ala Glu Ser Thr Val Tyr Val Ser Pro Arg His Gln ArgThr Gly Leu Gly Ser Thr Leu Tyr Thr His Leu Leu Lys Ser Leu Glu Ala GlnGly Phe Lys Ser Val Val Ala Val Ile Gly Leu Pro Asn Asp Pro Ser Val ArgMet His Glu Ala Leu Gly Tyr Ala Pro Arg Gly Met Leu Arg Ala Ala Gly PheLys His Gly Asn Trp His Asp Val Gly Phe Trp Gln Leu Asp Phe Ser Leu ProVal Pro Pro Arg Pro Val Leu Pro Val Thr Glu Ile

in which X is Met or Val, said DNA consisting of between 549 and 625nucleotides wherein X is encoded by ATG.
 2. The isolated DNA of claim 1,consisting of the nucleotide sequence (SEQ ID No. 2 from nucleotide 2 tonucleotide 549): NTG AGC CCA GAA CGA CGC CCG GCC GACATC CGC CGT GCC ACC GAG GCG GAC ATG CCG GCG GTC TGC ACC ATC GTC AAC CACTAC ATC GAG ACA AGC ACG GTC AAC TTC CGT ACC GAG CCG CAG GAA CCG CAG GAGTGG ACG GAC GAC CTC GTC CGT CTG CGG GAG CGC TAT CCC TGG CTC GTC GCC GAGGTG GAC GGC GAG GTC GCC GGC ATC GCC TAC GCG GGC CCC TGG AAG GCA CGC AACGCC TAC GAC TGG ACG GCC GAG TCG ACC GTG TAC GTC TCC CCC CGC CAC CAG CGGACG GGA CTG GGC TCC ACG CTC TAC ACC CAC CTG CTG AAG TCC CTG GAG GCA CAGGGC TTC AAG AGC GTG GTC GCT GTC ATC GGG CTG CCC AAC GAC CCG AGC GTG CGCATG CAC GAG GCG CTC GGA TAT GCC CCC CGC GGC ATG CTG CGG GCG GCC GGC TTCAAG CAC GGG AAC TGG CAT GAC GTG GGT TTC TGG CAG CTG GAC TTC AGC CTG CCGGTA CCG CCC CGT CCG GTC CTG CCC GTC ACC GAG ATC

in which N is A or G.